Bacterial cellulose (BC), a type of nanopolymer produced by Acetobacter xylinum is a nanostructured mate-rial with unique properties and wide applicability. However, a standard medium used for the cultivation of BC, the Hestrin–Schramm medium, is expensive and prevents wide scale extension of BC applications.In this research, a relatively low-cost culture media was successfully developed from wood hot waterextracts for the Acetobacter xylinus 23769 strain. Hot water extract (HWE) is a residual material originat-ing from pulp mills and lignocellulosic biorefineries and consists of mainly monomeric sugars, organicacids and organics. The effects of different pH (5, 6, 7 and 8) and temperatures (26, 28 and 30◦C) were alsoexamined in this research. There were no significant differences in the crystallinity and the recorded I˛fraction of cellulose produced between Hestrin–Schramm and the HWE medium. The maximum produc-tion of 0.15 g/l of BC was obtained at a pH of 8 and temperature of 28◦C. Glucose and xylose in the HWEwere the main nutrient sources utilized in all BC cultivations based on high-pressure liquid chromatog-raphy (HPLC) results. HWE was shown to be a suitable carbon source for BC production, and a processwas established for BC production from lignocellulosic feedstocks without using any modification of theHWE. HWE is an abundant and relatively inexpensive forest by-product. Using HWE for BC productioncould reduce burdens on the environment and also, achieve the goal of large scale BC production at lowcost without using added culture nutrients.